Long term objectives of this proposal are to begin mapping the 2', 3' cyclic nucleotide 3' phosphohydrolase (EC 3.1.4.27, CNP) gene, and provide the gene and cDNAs for further study. Its function is not yet known, but it is found at very high activities within the nervous system. In human diseases and disorders such as multiple sclerosis and SSPE in which CNS myelin is affected, this enzyme may be released from myelin membranes. This enzyme is also expressed in the outer rod segments within the visual system and is of interest there as well. Localization of this enzyme in adults brain and spinal cord is almost exclusively to the cell types that elaborate myelin, or in the case of the outer rod segments, rather extensive membrane reorganization, strongly suggesting that this enzyme is of importance and interest to our basic understanding of membrane biogenesis and maintenance within the nervous system. The proposed experiments utilize recombinant DNA to introduce human CNP cDNAs and altered cDNAs into mammalian cells. These cells will be transfected and the gene expression will be studied in transient and stable transfected HeLa S3 and in transiently transfected COS cells. this is being done to determine the location of the essential apparent 2',3'-cyclic nucleotide hydrolysis site on the enzyme. The gene will be mapped within Chromosome 17. To this end, we propose to use recently-characterized crossover DNA panels from large families for which information is available specifically on chromosome 17. Polymorphic site information and the development of an allele-specific assay now make these experiments feasible and timely. We will also isolate the human gene from a YACneo library, complete the DNA sequencing of the human gene and examine additional properties of the expressed gene in cell culture. Specific aims of this proposal are: 1. To isolate both CNP2 and CNP1 full-length cDNA clones from our own human corpus callosum cDNA library. 2. To insert the human gene DNA and cDNA into an SV40 plasmid-derived vector for expression of the CNP gene in cell culture. 3. To construct deletional mutants in the cloned human cDNAs to human CNP to determine the site of the apparent 2',3'-cyclic nucleotide binding region corresponding to the elastase fragment of the enzyme. 4. To map the human gene using DNA crossover family panels recently characterized on chromosome 17, the knowledge of the c -> t polymorphism at nucleotide 1215 within the most 3'-intron and our recent chromosomal assignment. 5. To compare the specific chromosomal localization of both the human and mouse CNP genes in somatic cell DNA hybrids by PCR. 6. To screen and clone the complete human CNP gene using a human genomic YACneo library using appropriate intron and exon primer pairs with PCR amplification as the primary screen and blotting and PCR as the secondary screen. 7. To continue sequencing the remainder of the human CNP gene and obtain additional important 5' sequence not currently available.